Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.
...
PMID:Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12. 351 53

Like the synthesis of alkaline phosphatase, the synthesis of outer membrane PhoE protein is shown to be dependent on the phoM gene product in phoR mutants of E. coli K12. This phoM gene has been cloned into the multi-copy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The gene was localized on the hybrid plasmids by analysis of deletion plasmids constructed in vitro and of mutant plasmids generated by gamma delta insertions. Interestingly, two of the selected hybrid plasmids contained the entire phoA-phoB-phoR region of the chromosome, as a multiple copy state of these genes results in the constitutive synthesis of alkaline phosphatase. The presence of multiple copies of the phoM gene hardly influences the level of expression of alkaline phosphatase and PhoE protein in a pho+ strain, but significantly increases the levels of these proteins in a phoR mutant strain.
...
PMID:Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12. 609 47

The SOS-function-inducing activities of 42 chemical mutagens were investigated in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ37 . To correct for the inhibitory effects of test chemicals on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Most of the mutagens reported to be mutagenic to the Ames' Salmonella tester strains showed the SOS-function-inducing activity. The inducible SOS repair may be responsible for not only base-change mutations but also frameshift mutations. However, 9-aminoacridine, ethidium bromide and 4-nitro-o-phenylenediamine did not induce the SOS function, suggesting that the mutagenesis induced by these mutagens may occur independently of SOS repair. Present results support the SOS mutagenesis model that error-prone SOS repair plays an important role in mutagenesis induced by most chemical mutagens.
...
PMID:The SOS-function-inducing activity of chemical mutagens in Escherichia coli. 620 35

The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG. Upstream to these sequences, there is a typical procaryotic promoter. TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.
...
PMID:The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli. 627 2

Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.
...
PMID:Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli. 628 49

The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established. The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension. This peptide has the general characteristics of a signal sequence. The promoter region of phoE has no homology with the consensus sequence of E. coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed. The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782. A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed. Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212. When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found. Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.
...
PMID:Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. 634 1

Most Escherichia coli K12 strains survive for a relatively long time outside the laboratory. Under the same conditions the isoallelic E. coli K12 relA mutants die faster because they lack the stringent response. The killing rate is increased by using a plasmid-encoded suicide system consisting of the phage T7 lysozyme gene driven by the E. coli alkaline phosphatase gene promoter (phoA). Cells containing this system were rapidly and effectively killed as soon as phosphate was made limiting. The combination of the chromosomal relA mutation and a conditional suicide system of this type provides an effective means of biological containment for recombinant E. coli strains.
...
PMID:Escherichia coli K12 relA strains as safe hosts for expression of recombinant DNA. 776 12

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.
...
PMID:Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli. 790 63

Tumour-specific isoenzymes and tumour markers in serum are potentially useful in the detection and monitoring of liver metastases. An experimental rat model was used in the search for such isoenzymes and to study factors affecting their serum levels. Splenic injection of CC531 colon carcinoma cells in syngeneic WagRij rats caused liver metastases after 3 weeks with concomitant and significant increases in serum levels of gamma-glutamyltransferase (GT) and alkaline phosphatase (ALP). The presence of tumour-specific isoforms of both enzymes, as well as increased amounts of the liver isoform of ALP, were demonstrated in serum. The serum levels of the tumour variants were clearly related to their elimination rates from the circulation. Thus, the slow clearance of the tumour ALP resulted in high serum levels of this isoform, compared with the more rapid elimination of tumour GT and its lower serum level. When using another colon carcinoma cell line (DHD/K12), metastatic to liver in BD IX rats, no increases in serum GT were detected. This was related to the rapid elimination from the circulation of the GT variant from the DHD/K12 metastatic tissue. The relatively high amount of the tumour ALP isoform detected in serum during growth of the CC531 liver metastases indicated that this isoform could be useful as a marker of tumour growth.
...
PMID:Serum gamma-glutamyltransferase and alkaline phosphatase during experimental liver metastases. Detection of tumour-specific isoforms and factors affecting their serum levels. 1002 18

The production and characterization of an anti-clenbuterol single-chain Fv antibody (CBLscFv)-bacterial alkaline phosphatase (AP) fusion protein are described. The CBLscFv and the phoA gene of Escherichia coli strain K12 chromosomal DNA were cloned by PCR and sequentially inserted into the expression vector pBV220 to express the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS. SDS-PAGE and western blot analyses revealed that the fusion protein showed a molecular weight of 73 kDa and bound with the antibacterial AP monoclonal antibody. Determination of enzymatic activity indicated that k(cat) and K(m) values of the fusion protein were 113.60 s(-1) and 29.82 microM, respectively. Competitive direct enzyme-linked immunosorbent assay based on the obtained fusion protein indicated that the average concentration required for 50% inhibition of binding (IC(50)) and the limit of detection for CBL were 4.74 +/- 0.003 (n = 3) and 0.54 +/- 0.004 (n = 3) microg/l, respectively, and the linear response range extended from 1.13 to 69.68 microg/l. Cross-reactivity studies showed that the fusion protein did not cross-react with CBL analogs. The present findings indicate that the production of the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggest that it could be further used to develop a one-step ELISA for the specific detection of CBL.
...
PMID:Production and characterization of a single-chain Fv antibody-alkaline phosphatase fusion protein specific for clenbuterol. 2008 89


<< Previous 1 2 3 Next >>

---